Absence of [3H]SCH 23390 binding sites in the rat adrenal gland

The binding of D2-dopamine receptor ligand [3H]spiperone and selective D1-ligand [3H]SCH 23390 to the rat adrenal gland and striatum has been compared. [3H]Spiperone showed specific binding in both tissues revealing a Bmax of 887 fmol mg-1 protein and KD of 0.38 nM, and B of 34 fmol mg-1 protein and KD of 0.66 nM in the striatum and adrenal gland, respectively. On the other hand, [3H]SCH 23390 showed a specific binding to the striatal tissue with Bmax of 747 fmol mg-1 protein and KD of 0.70 nM, while in the adrenal tissue no specific binding was observed. These results apparently indicated only D2-dopamine receptor binding sites being present in the rat adrenal gland.     

Characterization of [3H]zetidoline binding to rat striatal membranes

The binding of [3H]zetidoline, a novel neuroleptic agent, to rat brain striatal membranes was investigated in-vitro. The optimal binding conditions for [3H]zetidoline differed from those for [3H]spiperone in pH, temperature and time. [3H]Zetidoline has high affinity for striatal dopamine receptors. Its binding is saturable, stereo-specific, has a low non-specific component and is reversible and tissue specific. The Scatchard analysis gave a biphasic curve, indicating that [3H]zetidoline interacts with more than one population of receptor sites (B'max = 67 fmol mg-1 protein, K'd = 0.11 nM; B"max = 500 fmol mg-1 protein, K'd = 2.49 nM). Kinetic analysis of rates of association and dissociation yielded a Kd value in agreement with that measured at equilibrium. Inhibition studies indicated that only dopamine and dopaminergic agents are able to displace [3H]zetidoline from its binding sites, and in a different rank order from that for displacement of [3H]spiperone. (-)-Sulpiride was especially effective in inhibiting [3H]zetidoline specific binding. Furthermore, like that of [3H]benzamides, [3H]zetidoline binding appears to be highly Na+-dependent and Li+ only partially substitutes Na+.     

The effect of benzodiazepines on the binding of [3H]SCH 23390 in vivo.

The effect of flunitrazepam upon the binding of [3H]SCH 23390 in vivo was investigated. Acute treatment with flunitrazepam decreased the binding of [3H]SCH 23390 in the striatum in a dose-dependent manner. The time course of radioactivity in the striatum, cerebral cortex and cerebellum in controls and flunitrazepam (1 mg/kg)-treated mice was measured after intravenous injection of [3H]SCH 23390. The binding kinetics were calculated, using the cerebellum as a reference region for the estimation of the amount of free ligand in the brain. Flunitrazepam significantly decreased the input rate constant to the receptor compartment and the dissociation rate constant in vivo. An in vivo displacement study, using carrier SCH 23390, also showed significant reduction in the dissociation rate constant of [3H]SCH 23390 in vivo. The drug Ro 15-1788 reversed the effect of flunitrazepam, suggesting that this reduction in binding of [3H]SCH 23390 was mediated by benzodiazepine receptors. To evaluate the relationship between the reduction in binding of [3H]SCH 23390 in vivo and in vivo occupancy of benzodiazepine receptors, in vivo occupancy of benzodiazepine receptors was measured using [3H]Ro 15-1788. A non-linear relationship was found between the reduction in dopamine D1 receptor binding in vivo and the occupancy of benzodiazepine receptors in vivo, indicating that benzodiazepines exerted the maximum change in dopamine receptor binding at a low fractional occupancy of receptors.     

Binding of [3H]ADTN to rat striatal membranes

The conformationally restricted dopamine analogue ADTN binds in a specific saturable manner to rat striatal membranes. Analysis of the data suggests a single binding site. Binding of [3H]ADTN is displaced by a wide range of dopamine agonists and antagonists (both typical and atypical). The potency of ADTN derivatives to displace [3H]ADTN correlates well with their activity as agonists in other tests both in vivo and in vitro.     

The binding of [3H]N-(chloroethyl)-norapomorphine to rat striatal membranes

On the stretch-induced discharge activity of the isolated crayfish sensory neuron flurazepam (less than or equal to 3 X 10(-4) M) and Ro 15-1788 (less than or equal to 10(-3) M) produced reversible concentration-dependent excitation, but oxazepam only produced depression (less than or equal to 5 X 10(-4) M). Similar divergent effects on the membrane properties were observed. Oxazepam increased the threshold to firing without changing resting potential, membrane resistance or the GABA-mediated IPSP. In contrast flurazepam and Ro 15-1788 produced a concentration-dependent decrease in threshold. Flurazepam did not alter membrane resistance or resting potential but facilitated GABA transmission. Ro 15-1788 had the opposite effect on the GABA synapse, and also depolarized the resting potential but did not alter membrane resistance. The change in spike threshold appeared to be an important component in producing discharge excitation or depression. These results not only demonstrate the capability of the sensory neuron to discriminate between structures of benzodiazepines, but also that these agents can produce divergent effects on synaptic and non-synaptic properties of a single neuron.     

Chronic imipramine reduces [3H]SCH 23390 binding and DA-sensitive adenylate cyclase in the limbic system

[3H]SCH 23390 binding and dopamine (DA)-stimulated adenylate cyclase activity were measured in brain membrane preparations from rats chronically treated with imipramine (10 mg/kg twice daily for 14 days). [3H]SCH 23390 binding sites were decreased by 27% in the limbic system but only 14% in the striatum. The responsiveness of adenylate cyclase to DA was reduced by 38% in the limbic system but was not modified in the striatum. Concomitant treatment with alpha-methyltyrosine (alpha-MPT) (50 mg/kg daily for 14 days) prevented the imipramine-induced reduction in both [3H]SCH 23390 binding sites and the responsiveness of adenylate cyclase to DA.     

3H]SCH 23390 identifies D-1 binding sites in rat striatum and other brain areas

The specific in-vitro binding of [3H]SCH 23390 has been characterized and its use in the identification of D-1 sites in various brain regions examined. At a single ligand concentration (0.4 nM) the specific binding of [3H]SCH 23390 to striatal membranes was routinely 98% of total binding as defined using 10(-5) M cis-flupenthixol. Specific binding at 37 degrees C reached equilibrium at 15 min and was reversible with a t1/2 for dissociation of 14 min. Specific binding of [3H]SCH 23390 over a range of concentrations (0.01-3.5 nM) was saturable (Bmax 73 pmol g tissue-1) of high affinity (Kd 0.36 nM) and to a single population of binding sites. Specifically bound [3H]SCH 23390 (0.4 M) was stereo selectively displaced by the isomers of butaclamol and flupenthixol but not by the D-2 selective antagonist, sulpiride. 5-HT, noradrenaline and cinanserin caused little or no displacement. Specific binding of [3H]SCH 22390 (0.4 nM; as defined using 10(-5) M cis-flupenthixol) showed marked regional variation. Specific binding was highest in the striatum; high levels were also observed in the mesolimbic area and substantia nigra. Lower specific binding was found in the frontal cortex and superior colliculus with the lowest levels in cerebellar preparations. The inclusion of 3 X 10(-7) M cinanserin did not alter the extent of specific binding observed in any brain region. The properties of [3H]SCH 23390 suggest it to be an excellent ligand for identification of D-1 sites in a variety of brain regions.     

Chronic treatment with antidepressants decreases the number of [3H]SCH 23390 binding sites in the rat striatum and limbic system

Binding of [3H]SCH 23390 to dopamine D-1 receptors and of [3H]spiperone to D-2 sites was measured in identical membrane preparations of the striatum and limbic system of rats treated chronically (twice daily, for two weeks) with antidepressants. Chronic administration of imipramine, amitriptyline, mianserin, citalopram, bupropion, iprindole and electroconvulsive shocks, but not benztropine or cyproheptadine (non-antidepressants) decreased the number of [3H]SCH 23390 binding sites, while no change in the parameters of [3H]spiperone binding was observed. The serotonin2 receptor antagonist ketanserin when added to the incubation medium had no effect on [3H]SCH 23390 binding to D-1 sites. The results suggest that D-1 receptor subsensitivity is a component of the therapeutic effect of antidepressants.     

Comparison of the pharmacological characteristics of [3H]raclopride and [3H]SCH 23390 binding to dopamine receptors in vivo in mouse brain

In vivo binding of the benzamide derivative [3H]raclopride was studied in mouse brain. The binding was saturable, reversible and stereospecific. Non-specific binding was 5-15% of the total binding. Pharmacological characterization of the binding indicated labelling of dopamine D2 receptors since the binding was potently inhibited by compounds with high affinity for this receptor in vitro. On the other hand, compounds with low affinity in vitro i.e., dopamine D1-selective compounds were weak or inactive as inhibitors of [3H]raclopride binding. A comparison of the pharmacological characteristics of [3H]raclopride and [3H]SCH 23390 binding in vivo indicated that compounds with selectivity in vitro retained this selectivity in vivo. Thus, spiroperidol, haloperidol, 1-sulpiride, clebopride, LY 171555 and (-)-NPA ((-)-N-propyl-norapomorphine) were D2 selective while SCH 23390, SKF 38393 and SKF 75670 were D1 selective. Clozapine, tilozepine, cis-flupentixol, chlorpromazine and butaclamol were non-selective both in vitro and in vivo. However, a few compounds changed profile in vivo compared to in vitro. Thus, fluperlapine and fluphenazine had a dual D1-D2 receptor profile in vitro but were D1- or D2-selective in vivo, respectively. Pergolide and molindone which were D2-selective in vitro both had a dual D1-D2 receptor profile in vivo. In conclusion, [3H]raclopride, in vivo, selectively labels the dopamine D2 receptor. Comparison of the pharmacological characteristics of [3H]raclopride and [3H]SCH 23390 binding in vivo supported the that the dopamine D1 receptor is an important target for a variety of neuroleptics, especially of the clozapine type. This may indicate that blockade of the dopamine D1 receptor conveys antipsychotic action.     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.     

Binding of [3H]SCH 23390 to dopamine D-1 receptors in rat retina in vitro

[3H]SCH 23390 binding was examined in membranes of rat retina. The binding was saturable with a dissociation constant of 0.2 nM and the maximum number of binding sites was 236 +/- 74 fmol/mg protein. The pharmacology of [3H]SCH 23390 binding indicated that the binding was specific for a dopamine D-1 receptor because the binding was preferentially inhibited by D-1-selective agonists and antagonists but not by dopamine D-2-selective agonists and antagonists. The same membrane preparations were used to characterize the dopamine D-2 receptor binding as measured with [3H]spiperone and the amount of binding sites was found to be similar to the amount of D-1 sites. It is concluded that [3H]SCH 23390 is a useful tool to examine dopamine D-1 receptors in the retina.     

A study on the localization of [H]sulpiride binding sites in rat striatal membranes

The specific binding of [³H]sulpiride to purified rat striatal synaptic membranes has been examined. This binding component was unchanged in rats with 6-hydroxydopamine-induced lesions but was significantly reduced (36%) in 24 hr kainate-lesioned, and (53%) in rats with cortical ablations (28 days postoperative). These results suggest that specific [³H]sulpiride binding sites are present on both slriatal interneurones and on the cortico-striatal pathway. This conclusion is inconsistent with the present classification of D₁ and D₂ receptors.     

The characterization of [3H]sulpiride binding sites in pig striatal membranes

Pig striatal membranes have [3H]sulpiride-binding sites similar to those identified in rat striatal membranes. The pharmacological profile indicates that this binding is to dopamine receptors. Agonist displacement of [3H]sulpiride binding in pig striatal membranes is subject to guanine nucleotide regulation. This effect is mimicked by heat treatment. N-ethyl maleamide (20 microM) and dithioerythritol (3 mM) decrease agonist affinity for the [3H]sulpiride-binding site in pig striatal membranes without significantly affecting maximal displacement.     

3H]SCH 23390 labels dopamine D-1 receptor sites in the rat kidney

The binding of [3H]SCH 23390, a selective dopamine D-1 receptor radioligand, to rat kidney cortical membranes was studied. Scatchard analysis revealed a single class of binding sites. Of dopamine, noradrenaline and serotonin, dopamine was the most potent of these to displace [3H]SCH 23390 binding. The selective D-1 ligands SCH 23390 and SK & F 38393 were more potent to displace [3H]SCH 23390 than the selective D-2 ligands S-sulpiride and LY 171555, thus indicating that [3H]SCH 23390 binds predominantly to dopamine D-1 receptor sites.     

Acetylcholinesterase potentiates [3H]fluorowillardiine and [3H]AMPA binding to rat cortical membranes

In addition to its action at cholinergic synapses acetylcholinesterase (AChE) has been proposed to modulate neuronal activity by mechanisms unrelated to the hydrolysis of acetylcholine. We have investigated the effects of AChE on the binding of the specific AMPA receptor agonists (S)-[3H]5-fluorowillardiine ([3H]FW) and [3H]AMPA to rat cortical membranes. Pretreatment of membranes with AChE causes a dose-dependent increase in the binding of both radiolabelled agonists with a maximal increase to approximately 60% above control. This increase is completely blocked by the specific AChE inhibitors propidium, physostigmine, DFP and BW 284C51. AChE pretreatment had no effect on [3H]kainate binding. [3H]FW binding to membranes from young (15-day-old) rats is four orders of magnitude more sensitive to AChE modulation than membranes from adult rats (EC50 values of 4x10(-5) and 0.1 unit/ml, respectively) although the total percentage increase in binding is similar. Furthermore, the AChE-induced potentiation of [3H]FW binding is Ca2+ - and temperature-dependent suggesting an enzymatic action for AChE in this system. Saturation binding experiments with [3H]FW to adult membranes reveal high and low affinity binding sites and demonstrate that the main action of AChE is to increase the Bmax of both sites. These findings suggest that modulation of AMPA receptors could provide a molecular mechanism of action for the previously reported effects of AChE in synapse formation, synaptic plasticity and neurodegeneration.